The objective of this proposal is to develop, validate and apply methods for measuring DNA damage as biological markers of carcinogen exposure in chewers of betel quid (BQ) with or without tobacco (T). Three approaches will be used: (a) in vitro modification of DNA by BQ/T constituents, (b) detection of DNA damage and toxic effects in cultured human buccal epithelial cells and explants treated as in (a), and (c) measurement of DNA damage in exfoliated buccal epithelial cells and buccal mucosa biopsies from exposed human subjects. For (c) specimens will be collected from about 50 subjects in each of four habit groups: (i) chewers of BQ with T, (ii) chewers of BQ only; (iii) chewers of T only and (iv) subjects with no chewing and smoking habits. The following assays for DNA-damage will be studied: 1) Assays for DNA strand breaks: Fluorimetric analysis of DNA unwinding in buccal epithelial cells exposed in vitro and in vivo to BQ/T. 2) Assays for oxidative DNA base damage: Quantification of thymine glycol, and 8-hydroxyguanine levels by HPLC with electrochemical detection or by GC-MS. 3) Immunoassays for carcinogen-DNA adducts: Detection and estimation of O6- alkyldeoxyguanine residues by RIA using available poly- or monoclonal antibodies (after HPLC clean-up), immuno slot-blot techniques and immunocytological assays. 4) Detection of carcinogen-DNA adducts by 32P-postlabelling: Randerath's method with modification will be employed to detect the in vitro and in vivo modification of DNA by BQ/T constituents. 5) Structural identification of DNA base adducts: They will be identified/confirmed after formation of electron-capturing derivatives by NICI-MS and by GC-ECD comparisons with authentic carcinogen adducts. Immunoassays for relevant adducts will then be developed. This study should provide a tissue dosimeter of carcinogen exposure through the quantitation of several types of DNA adducts/modifications produced by BQ/T in human buccal epithelial cells. As such DNA lesions can now be detected with high sensitivity, their relationship to the duration and frequency of use of BQ/T and to cancer risk can be examined in molecular/epidemiological studies. By comparing DNA-base damage in the study groups i-iv, the major cancer-causing ingredients in BQ/T should be identified, and the relative contributions of tobacco and areca nut-related compounds to betel quid associated cancers should be discerned. The methodology developed should also be applicable to snuff dippers and tobacco chewers in western countries.